Vesicular transport shuttles proteins and membranes among the different organellar compartments within the cell. Coat proteins act as the core machinery that initiates vesicle formation. Coat Protein I (COPI) has been a paradigm to studying this process, and has been shown to be regulated by the small GTPase ADP- Ribosylation Factor 1 (ARF1). In the last funding period, we have made significant advancements in understanding COPI-dependent transport. First, we note that intracellular transport is now also appreciated to occur through tubular transport, but the core machinery responsible for tubule formation has been unknown. Addressing this issue, we have found that the COPI complex also initiates tubule formation, which is involved in promoting anterograde intra-Golgi transport and Golgi ribbon formation. As such, we propose in the current renewal to identify tethers predicted to act in the docking of COPI tubules to target compartment, and also determine how cargoes are transported in these tubules. Second, we note that the GTPase-activating protein (GAP) that deactivates ARF1, ARFGAP1, has been found previously to have a novel role, acting also as a coat component. However, there has been continuing controversy regarding this role, in particular from COPI reconstitution studies that have used liposomes rather than Golgi membrane. Thus, we propose to elucidate why the liposome-based system has not been able to reconstitute the coat function of ARFGAP1. Moreover, we will clarify the role of this GAP with respect to ARFGAP2/3, which are two closely related members. Third, we have recently identified a novel mechanism of regulating COPI vesicle formation that involves a dehydrogenase reaction, and propose to further dissect out how this regulation is achieved. Fourth, we have also recently elucidated the complexity by which a critical lipid, phosphatidic acid (PA), acts in COPI vesicle fission. Thus, we will also seek a better mechanistic understanding of how PA acts in this process. Altogether, these proposed investigations will likely advance not only a mechanistic understanding of COPI transport, but also general principles that underline intracellular transport. Moreover, because this basic process is critical for many cellular events, our results may also suggest new molecular insights into how multiple human diseases occur. PUBLIC HEALTH RELEVANCE: We study how proteins and membrane are transported within the cell. We anticipate that our proposed studies will advance a fundamental understanding of this transport. Moreover, because this process is critical for multiple aspects of cellular function, we further anticipate in shedding key molecular insights into pathologic events that result in human diseases.